Urinary tract infection (UTI) is the most frequently diagnosed kidney and urologic disease and Escherichia coli is by far its most common etiologic agent. Individuals at high risk include neonates, preschool girls, sexually active women, and elderly women and men. Half of all women experience at least one UTI, leading to 6.8 million annual physician visits, 1.3 million emergency room visits, and 245,000 hospitalizations with annual costs of $2.4 billion. Emergence of antibiotic resistance, allergic reaction to pharmaceuticals, alteration of normal gut flora, and failure to prevent recurrent infections document the need for effective prevention. Vaccination has been approached by several groups without marked or long lasting protection. Here, we present a plan to systematically identify surface-exposed proteins that are synthesized in vivo and are specific to uropathogenic strains of E. coli. Surface-exposed proteins will be determined by: screening for outer membrane proteins that react with sera from chronically infected mice; predicting surface-expressed proteins bioinformatically; and releasing surface-expressed domains by limited proteolysis. Candidate antigens that meet these sequential screens will be used for vaccination in the established murine model of urinary tract infection. As Specific Aims, we propose to: 1) Identify surface- exposed proteins that are synthesized in vivo and conserved among uropathogenic E. coli strains; and 2) Determine the efficacy of candidate proteins as components of a multivalent subunit vaccine to protect against urinary tract infection. Our goal will be to include the most protective antigens in a multivalent subunit vaccine for prevention of UTI by uropathogenic E. coli. PUBLIC HEALTH RELEVANCE: Urinary tract infection (UTI) is the most frequently diagnosed kidney and urologic disease and Escherichia coli is by far its most common etiologic agent. Individuals at high risk include neonates, preschool girls, sexually active women, and elderly women and men. Half of all women experience at least one UTI, leading to 6.8 million annual physician visits, 1.3 million emergency room visits, and 245,000 hospitalizations with annual costs of $2.4 billion. Emergence of antibiotic resistance, allergic reaction to pharmaceuticals, alteration of normal gut flora, and failure to prevent recurrent infections document the need for effective prevention. Vaccination has been approached by several groups without marked or long lasting protection. Here, we present a plan to systematically identify surface-exposed proteins that are synthesized in vivo and are specific to uropathogenic strains of E. coli for use in a vaccine to prevent urinary tract infection.